Methods and materials for hematoendothelial differentiation of human pluripotent stem cells under defined conditions

ABSTRACT

Methods, kits and compositions for differentiating pluripotent stem cells into cells of endothelial and hematopoietic lineages are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application No. U.S.patent application Ser. No. 15/917,504 filed on Mar. 9, 2018, which is acontinuation of U.S. patent application Ser. No. 14/206,778, filed Mar.12, 2014, now U.S. Pat. No. 9,938,499, granted Apr. 10, 2018, whichclaims the benefit of U.S. Provisional Application No. 61/779,564 filedon Mar. 13, 2013, the contents of which are incorporated by reference intheir entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

This invention was made with government support under Grants No.HL099773 and HL116221 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The advent of human pluripotent stem cell technologies has provided theopportunity to produce endothelial and hematopoietic cells in vitro forfunctional studies and therapies. Previously, co-culture systems usingthe mouse bone marrow stromal cell line, OP9, have been used toestablish efficient and scalable differentiation of human pluripotentstem cells (hPSCs) into endothelial and blood lineages. However,co-culture systems that rely on mouse feeder cells and serum (i.e.,xenogenic sources) have limited utility for studying hPSC response tospecific growth factors. Moreover, such systems have further limitationswhen considered in the context of manufacturing clinical gradetherapeutic blood cells.

In light of the shortcomings of prior culture systems, new culturesystems are needed to provide sources of endothelial and blood lineagesthat are suitable for use in clinical settings without the risk ofintroduction of xenogenic contamination.

SUMMARY OF THE INVENTION

In one aspect provided herein is a method for differentiating humanpluripotent stem cells comprising: (a) providing human pluripotent stemcells; and (b) culturing the human pluripotent stem cells under hypoxicconditions in a cell culture medium comprising FGF2, BMP4, Activin A,and LiCl for a period of about two days to form a cell population of^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells withmesenchymoangioblast potential.

In some embodiments the method also includes the step of (c) exposingcells at the primitive mesoderm stage of step (b) to a mixturecomprising components FGF2 and VEGF under hypoxic conditions for aperiod of about 1-2 days to obtain a population comprising^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm with hemangioblast(HB-CFC) potential and hematovascular mesoderm cells(^(EMH)lin⁻KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) with a potential to formhematoendothelial clusters when cultured on OP9 cells. In someembodiments, the method further includes the step of: (d) exposing thecells at the hematovascular mesoderm stage of step (c) to a mixturecomprising components FGF2, VEGF, IL6, SCF, TPO, and IL3 for about oneday to achieve formation of CD144⁺CD73⁺CD235a/CD43⁻ non-hemogenicendothelial progenitors (non-HEP), CD144⁺CD73⁻CD235a/CD43⁻ hemogenicendothelial progenitors (HEPs), CD144⁺CD73⁻CD235a/CD43⁺41a⁻ angiogenichematopoietic progenitors (AHP), and CD43⁺CD41a⁺ hematopoieticprogenitor cells. In further embodiments, the method also comprises thestep of: (e) continuing to expose the HEPs and emerging hematopoieticprogenitor cells to a mixture of FGF2, VEGF, IL6, SCF, TPO, IL3 undernormoxia for about three days resulting in hematopoietic expansion toobtain a population of CD43⁺ hematopoietic progenitors composed ofCD43⁺CD235a⁺CD41a⁺ erythromegakaryocytic progenitors andlin⁻CD34⁺CD43⁺CD45^(+/−) multipotent hematopoietic progenitors.

In some embodiments the mixture to be used in any of the precedingmethods consists essentially of the mentioned components. In someembodiments the mixture to be used is xenogen-free.

In some embodiments the human pluripotente stem cells are provided on asubstrate treated with Tenascin-C.

In a further aspect provided herein is a xenogen-free culture medium fordifferentiating human pluripotent stem cells, comprising IF9S mediumsupplemented with: about 50 to about 250 ng/ml BMP4; about 10 to about15 ng/ml Activin A; about 10 to about 50 ng/ml FGF2; and about 1 mM toabout 2 mM LiCl.

In a related aspect provided herein is a xenogen-free culture medium fordifferentiating human pluripotent stem cells, comprising IF9S mediumsupplemented with: about 10 to about 50 ng/ml FGF2; and about 20 toabout 50 ng/ml VEGF. In some embodiments, where the medium contains FGF2and VEGF, the medium also includes a hematopoietic cytokine. In someembodiments, the hematopoietic cytokine comprises: about 50 to about 100ng/ml SCF; about 50 to about 100 ng/ml TPO; about 50 to about 100 ng/mlIL-6; and about 5 to about 15 ng/ml IL-3. In some embodiments, any ofthe foregoing media consist essentially of the IF9S medium and thesupplemented components. In some embodiments, any of the foregoing mediaare provided in a concentrated form.

In another aspect provided herein is a xenogen-free cell culture systemfor differentiating human pluripotent stem cells into mesoderm,endothelial, and hematopoietic progenitor cells, comprising: humanpluripotent stem cells seeded as a single cell suspension on a substratecomprising a layer of Tenascin C at a concentration of at least about0.25 μg/cm² to 1 μg/cm²; and a xenogen-free culture medium comprisingIF9S medium supplemented with: about 50 to about 250 ng/ml BMP4; about10 to about 15 ng/ml Activin A; about 10 to about 50 ng/ml FGF2; andabout 1 to about 2 mM LiCl. In some embodiments the xenogen-free culturemedium to be used in the xenogen-free cell culture system furthercomprises hematopoietic cytokines. In some embodiments the hematopoieticcytokines in the xenogen free medium comprise about 50 to about 100ng/ml SCF, about 50 to about 100 ng/ml TPO, about 50 to about 100 ng/mlIL-6, and about 5 to about 15 ng/ml IL-3. In some embodiments the layerof Tenascin C is at a concentration of about 0.5 μg/cm².

In another aspect provided herein is a method of differentiatingpluripotent stem cells, comprising the steps of: (a) providing humanpluripotent stem cells; (b) seeding the cells as a single cellsuspension on a substrate treated with Tenascin C; and (c) culturing theseeded cells in IF9S medium supplemented with BMP4, Activin A, FGF2, andLiCl under hypoxic conditions for a period of about two days to obtainabout 30% ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells withmesenchymoangioblast potential.

In some embodiments, the above method further comprises the step ofculturing the cells at ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitivemesoderm stage in IF9S medium supplemented with FGF2 and VEGF underhypoxic conditions for about 1-2 days to obtain^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm with hemangioblast(HB-CFC) potential and ^(EMH)linKDR^(hi)APLNR⁺PDGFRalpha^(lo/−)hematovascular mesodermal precursors with a potential to formhematoendothelial clusters when cultured on OP9 cells.

In a further aspect provided herein are purified populations of cellsgenerated by any of the foregoing methods for differentiating humanpluripotent stem cells, wherein the cells have not been exposed tonon-human constituents.

In a related aspect provided herein is a purified population of cellscreated by the methods described herein, wherein the cells are greaterthan about 35% ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cellswith a potential to form mesenchymoangioblast colonies.

In another aspect provided herein is a purified population of cells,wherein the cells are greater than about 35% of^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm with hemangioblast(HB-CFC) potential and 30% ^(EMH)lin⁻KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)hematovascular mesodermal cells with a potential to formhematoendothelial clusters when cultured on OP9 cells.

In a further aspect provided herein is a purified population of cells,wherein the population includes greater than about 40% of CD144⁺ cellscomprising CD144⁺CD73⁻CD235a/43⁻ hemogenic endothelial progenitors(HEPs), CD144⁺CD73⁻CD235a/CD43⁺CD41a⁻ angiogenic hematopoieticprogenitors, and CD144⁺CD73⁺CD235a/43⁻ non-hemogenic endothelialprogenitors (non-HEPs).

In another aspect provided herein is a population of cells, wherein thepopulation includes greater than about 30% CD43⁺ hematopoieticprogenitor cells composed of CD43⁺CD235a+CD41a⁺ erythromegakaryocyticprogenitors and lin⁻CD34⁺CD43⁺CD45^(+/−) multipotent hematopoieticprogenitors.

In a further aspect provided herein is a method of producingmesenchymoangioblasts, comprising the steps of: (a) providing humanpluripotent stem cells; (b) seeding the cells on a substrate treatedwith an effective amount of collagen; and (c) exposing the stem cells toa mixture comprising FGF2, BMP4, Activin A, and LiCl under hypoxicconditions for a period of about two days to form a population of^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells withmesenchymoangioblast potential. In some embodiments the collagen to beused comprises Collagen IV.

In yet another aspect provided herein is a cell culture medium fordifferentiating human pluripotent stem cells, comprising: 64 mg/LL-Ascorbic Acid 2-Phosphate Mg²⁺ salt, 40 μl/L monothioglycerol, 8.4μg/L additional sodium selenite, 10 mg/L polyvinyl alcohol, 1× GLUTAMAX,1× non-essential amino acids, 0.1× chemically-defined lipid concentrate,10.6 mg/L Holo-Transferrin, and 20 mg/L insulin.

In a further aspect described herein is a method for differentiatinghuman pluripotent stem cells comprising: (a) providing human pluripotentstem cells; and (b) culturing the human pluripotent stem cells underhypoxic conditions in a cell culture medium comprising FGF2, BMP4,Activin A, and LiCl for a period of about two days to form a cellpopulation of ^(EMH)lin−KDR+APLNR+PDGFRalpha+ primitive mesoderm cellswith mesenchymoangioblast potential.

In some embodiments the human pluripotent stem cells are cultured onTenascin C. In some embodiments the cell culture medium comprises anIF9S cell culture medium. In some embodiments the concentration, in thecell culture medium, of: BMP4 is about 50 ng/ml to about 250 mg/ml;Activin A is about 10 ng/ml to about 15 ng/ml; FGF2 is about 10 ng/ml toabout 50 ng/ml; and LiCl is about 1 mM to about 2 mM.

In some embodiments the method further comprises (c) culturing, underhypoxic conditions, the cell population obtained in step (b) in a cellculture medium comprising FGF2 and VEGF for a period of about 1-2 daysto obtain a cell population comprising ^(EMH)lin−KDR+APLNR+PDGFRalpha+primitive mesoderm with hemangioblast (HB-CFC) potential andhematovascular mesoderm cells(^(EMH)lin−KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) with a potential to formhematoendothelial clusters when cultured on OP9 cells. In someembodiments the concentration, in the cell culture medium, of FGF2 isabout 10 ng/ml to about 50 ng/ml; and VEGF is about 20 ng/ml to about 50ng/ml.

In some embodiments the method further comprises (d) culturing thehematovascular mesoderm cells of step (c), under hypoxic conditions, ina cell culture medium comprising FGF2, VEGF, IL6, SCF, TPO, and IL3 forabout one day to obtain a cell population comprisingCD144+CD73+CD235a/CD43− non-hemogenic endothelial progenitors (non-HEP),CD144+CD73−CD235a/CD43− hemogenic endothelial progenitors (HEPs),CD144+CD73−CD235a/CD43+41a− angiogenic hematopoietic progenitors (AHP),and CD43+CD41a+ hematopoietic progenitor cells. In some embodiments theconcentration, in the cell culture medium, of: FGF2 is about 10 ng/ml toabout 50 ng/ml; VEGF is about 20 ng/ml to about 50 ng/ml; SCF is about50 ng/ml to about 100 ng/ml; TPO is about 50 ng/ml to about 100 ng/ml;IL-6 is about 50 ng/ml to about 100 ng/ml, and IL-3 is about 5 ng/ml toabout 15 ng/ml.

In some embodiments the method further comprises (e) culturing, undernormoxia, the HEPs and hematopoietic progenitor cells in a culturemedium comprising FGF2, VEGF, IL6, SCF, TPO, IL3 for about three days toobtain an expanded population of CD43+ hematopoietic progenitorscomprising CD43⁺CD235a⁺CD41a⁺ erythromegakaryocytic progenitors andlin−CD34⁺CD43⁺CD45^(+/−) multipotent hematopoietic progenitors.

In some embodiments the method further comprises further coculturing theexpanded population of CD34+CD43+ hematopoietic progenitors for a periodof about three weeks on OP9 cells overexpressing DLL4 to obtain a cellpopulation comprising CD4+CD8+ double positive T cells, wherein thehuman pluripotent stem cells of step (b) are cultured on a Tenascin Csubstrate.

In a related aspect provided herein is a method of differentiating humanpluripotent stem cells, comprising at least one of: (i) culturing humanpluripotent stem cells under hypoxic conditions in a cell culture mediumcomprising FGF2, BMP4, Activin A, and LiCl for a period of about twodays to form a cell population of ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺primitive mesoderm cells with mesenchymoangioblast potential; (ii)culturing, under hypoxic conditions, ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺primitive mesoderm cells with mesenchymoangioblast potential in a cellculture medium comprising FGF2 and VEGF for a period of about 1-2 daysto obtain a cell population comprising ^(EMH)lin−KDR+APLNR+PDGFRalpha+primitive mesoderm with hemangioblast (HB-CFC) potential andhematovascular mesoderm cells(^(EMH)lin⁻KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) enriched in cells with apotential to form hematoendothelial clusters when cultured on OP9 cells;(iii) culturing hematovascular mesoderm cells(^(EMH)lin⁻KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) cells, under hypoxicconditions, in a cell culture medium comprising FGF2, VEGF, IL6, SCF,TPO, and IL3 for about one day to achieve formation ofCD144+CD73+CD235a/CD43− non-hemogenic endothelial progenitors (non-HEP),CD144+CD73−CD235a/CD43− hemogenic endothelial progenitors (HEPs),CD144+CD73−CD235a/CD43+41a− angiogenic hematopoietic progenitors (AHP),and CD43+CD41a+ hematopoietic progenitor cells; (iv) culturing, undernormoxia, hemogenic endothelial progenitors (HEPs) and CD43+CD41a+hematopoietic progenitor cells in a culture medium comprising FGF2,VEGF, IL6, SCF, TPO, IL3 for about three days to obtain an expandedpopulation of CD43+ hematopoietic progenitors comprisingCD43+CD235a+CD41a+ erythromegakaryocytic progenitors andlin−CD34+CD43+CD45+/− multipotent hematopoietic progenitors; and (v)coculturing CD34+CD43+ hematopoietic progenitors for a period of aboutthree weeks on OP9 cells overexpressing DLL4 to obtain a cell populationcomprising CD4+CD8+ T cells.

In another aspect provided herein is a cell culture medium suitable forhematoendothelial differentiation of human pluripotent stem cells,comprising a base medium, L-ascorbic acid 2-phosphate Mg2+ salt,monothioglycerol, additional sodium selenite, polyvinyl alcohol,Glutamax™, non-essential amino acids (NEAA), chemically defined lipidconcentrate, Holo-Transferrin, and insulin.

In some embodiments the cell culture medium further comprises BMP4,Activin A, FGF2, and LiCl.

In other embodiments the culture medium further comprises FGF2 and VEGF.

In other embodiments the culture medium further comprises FGF2, VEGF,SCF, TPO, IL-6, and IL-3.

In some embodiments the cell culture medium comprises an IF9S medium. Insome embodiments the IF9S cell culture medium has the IF9S cell culturemedium formulation of Table 2.

In a related aspect provided herein is a 9S concentrated mediumsupplement, wherein dilution of the 9S concentrated medium supplement inan IMDM/F12 base medium yields an IF9S cell culture medium. In oneembodiment is a kit comprising the 9S concentrated medium supplement,and one or more of BMP4, Activin A, FGF2, LiCl, SCF, TPO, IL-6, IL-3,and Tenascin C.

In a further aspect provided herein is a defined cell culture system forhematoendothelial differentiation of human pluripotent stem cells,comprising an IF9S cell culture medium and a Tenascin C substrate foradherent growth of human pluripotent stem cells or their differentiatedprogeny along the hematoendothelial lineage. In some embodiments theIF9S cell culture medium is maintained under hypoxic conditions. In someembodiments the defined cell culture system further comprises humanpluripotent stem cells grown on the Tenascin C substrate.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, and patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 Schematic diagram of hematopoietic differentiation. The diagramshows hematopoietic development pathways, specific markers andfunctional assays used to identify each stage of development, andconditions used for hPSC differentiation in chemically defined medium.Main cell subsets observed in prior differentiation studies usingcoculture with OP9 feeders and current chemically defined cultures areshown.

FIGS. 2A-2B Identification of a unique molecular signature of overgrownOP9 stromal cells. (a) Venn diagram showing the overlap betweendifferentially expressed genes in overgrown OP9 day 8 versus freshlyconfluent OP9 day 4, and MS5 and S17. 21 genes marked with graybackground are uniquely overexpressed in day 8 OP9 cells as compare toall other tested cell lines. (b) Heat map of differentially expressedoverlapping genes as shown in (a). Tenascin-C (Tnc) is one of the topdifferentially overexpressed genes in over-confluent OP9 cells.

FIGS. 3A-3D Mesodermal development in hESC cultures differentiated onColIV vs TenC for 2, 3, and 4 days in chemically defined conditions. (a)Flow cytometry plots and graphs comparing percentage of^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ (A+P+) primitive mesodermal populationon days 2 and 3. (b) Flow cytometry plots and graphs comparingpercentage of KDR^(hi)CD31⁻ (HVMP), CD31⁺, and KDR^(lo)CD31⁻ populationson day 4. (c) Comparison of MB/HB-colony forming potential of day 2, day3, and day 4 cultures. (d) Hematopoietic and endothelial potentials ofKDR^(hi)CD31⁻ and KDR^(lo)CD31⁻ cells isolated from day 4 cellsdifferentiated in chemically defined conditions after coculture with OP9for 7 days. Upper panels show flow cytometry of TRA-1-85+ gated humancells and lower panels shows immunofluorescence staining of cells fromOP9 cocultures with KDR^(lo)CD31⁻ and KDR^(lo)CD31⁻ cells. (a), (b), and(c) bars are mean+SE from 3 experiments (*p<0.01).

FIGS. 4A-4D Development of endothelial progenitors in culturesdifferentiated on ColIV or TenC for 5 days in chemically definedconditions. (a) Flow cytometric analysis demonstrates major subsets ofVE-cadherin+ (VEC; CD144+) progenitors generated after 5 days of hESCculture in chemically defined conditions on ColIV and TenC. (b)Percentages of VEC+ (CD144+) cells and subsets generated in ColIV andTenC cultures. Error bars are mean+SE from 3 experiments (*p<0.01). (c)CFC potential of isolated VEC+ (CD144+) subset in serum-free clonogenicmedium with FGF2 and hematopoietic cytokines. (d) Endothelial andhematopoietic potential of day 5 VEC+ (CD144+) subsets. Progenitorsubsets sorted and cultured in either endothelial conditions withsubsequent tube formation assay, or on OP9 with immunofluorescent andflow cytometry results after 7 days. Scale bars, 100 μm.

FIGS. 5A-5D Development of hematopoietic progenitors in culturesdifferentiated on ColIV for 8 days in chemically defined conditions. (a)Flow cytometric analysis shows major subsets of CD43+ cells generated incultures on ColIV and TenC. (b) Cultures on TenC produce significantlymore CD43+ cells across 3 experiments (*p<0.01). (c) Hematopoietic-CFCpotential in serum-containing media is limited to the CD43+subpopulations. (d) Cultures differentiated on TenC have higher CFCpotential than cultures differentiated on ColIV, statisticallysignificant across 3 experiments (*p<0.01).

FIGS. 6A-6C T cell potential of CD43+ cells collected from H1 hESCsdifferentiated for 9 days in chemically defined conditions on ColIV andTenC. (a) Flow cytometric analysis of cells collected from ColIV andTenC conditions after culture on OP9DLL4 for 3 weeks. (b and c) Analysisfor T cell receptor rearrangement by genomic PCR. H1 T-Cell is theT-cells derived from differentiating H1; PB control is Peripheral Bloodpositive control; H1 hESC is undifferentiated H1 hESCs.

FIG. 7 The effect TGFβ inhibitor on hematopoietic development from H1hESCs in chemically defined conditions. Representative dot plotscollected from flow cytometry of day 3, 4, 5, and 8 of differentiationafter adding the TGFβ inhibitor, SB-431542 from day 2 to day 4 only. Onday 3, SB-431542 decreases PDGFRalpha expression, but increasesendothelial progenitors by days 4 and 5. By day 8, there is asignificant increase in CD43+ hematopoietic progenitors.

FIG. 8 Generation of KDR^(hi)CD31⁺ hematoendothelial progenitors incultures using different basal media and matrix protein. TeSR1 basemedium is TeSR1 without cytokines. DF4S is DMEM/F12-based mediasupplemented with 4 supplements; 64 mg/L L-ascorbic Acid 2-PhosphateMg²⁺ salt, 8.4 μg/L sodium selenite, 10.6 mg/L Holo-Transferrin, and 20mg/L Insulin. I4S is DF4S with IMDM-based media instead ofDMEM/F12-based media, but with the four previously mentionedsupplements. IF4S is DF4S with IMDM/F12-based media instead ofDMEM/F12-based media, but with the 4 previously mentioned supplements.VTN is vitronectin matrix; MTG is Matrigel® substrate; ColIV is CollagenIV matrix. Flow cytometry plots show percent of KDR^(hi)CD31⁺endothelial precursors of day 4 cells differentiated in each mediasupplemented with 50 ng/ml FGF2, BMP4 and VEGF in hypoxic conditions.

FIG. 9 Hematopoietic differentiation of iPSCs and 119 hESCs inchemically defined conditions. Top panel represents the number of cellsgenerated in cultures starting from day −1 when cells are plated oneither TenC or ColIV, up to day 9 of differentiation. The numbers ofCD31⁺ and CD43⁺ cells were calculated based on total number of cellstimes the percentage of positive cells based on flow cytometry. Thebottom panel displays dot plots of the percentage of CD43⁺ cells andtheir subsets of 19-9-7T human fibroblast iPSC line, BM19-9 human bonemarrow-derived iPSC line, and H9 human ESC line differentiated for eightdays on either ColIV or TenC.

FIG. 10 is a diagram of a hematopoietic differentiation timelinecomparing the efficiency of differentiation on Collagen IV vs. TenascinC.

DETAILED DESCRIPTION

Human pluripotent stem cell (hPSC) technologies provide the opportunityto study human development in vitro and develop patient-specific bloodcells without relying on HLA-matched donors. Previously, an efficientprotocol for the differentiation of hematopoietic stem/progenitor cellsusing a coculture method on the mouse stromal cell line, OP9, wasdeveloped (Vodyanik, et al., 2005; Vodyanik, et al., 2006). This systemreproduces primitive and definitive waves of hematopoiesis and can beused to obtain lin⁻CD34⁺CD38⁻CD45RA⁻CD90⁺CD117⁺CD43⁺CD45^(−/+)multipotent definitive hematopoietic progenitors with HSC phenotype andlymphoid cells, including T and B cells (Carpenter, et al., 2011;Kutlesa, et al., 2009; Schmitt and Zuniga-Pflucker, 2002; Vodyanik, etal., 2005).

Human pluripotent stem cell coculture with OP9 cells inducesmesendodermal and hemogenic endothelial differentiation. Upon platinghPSCs onto OP9 cells, the hPSCs begin to express the mesodermal markerapelin receptor (APLNR), VEGFR2 (KDR), and PDGFRalpha and acquiremesenchymoangioblast (MB) and hemangioblast (HB) potential (days 2-3 ofdifferentiation). With advanced maturation, KDR⁺APLNR⁺ mesodermal cellsupregulate KDR expression and downregulate PDGFRα, which is enriched incells with the potential to form hematoendothelial clusters whencultured on OP9 cells.

At day 2-3.5 of differentiation, KDR⁺APLNR⁺ cells lack the typicalEndothelial (CD31, VE-cadherin (CD144)), endothelial/Mesenchymal (CD73,CD105) and Hematopoietic (CD43, CD45) markers, i.e. have an ^(EMH)lin⁻phenotype. ^(EMH)lin⁻ cells lack endothelial (CD31 and CD144),mesenchymal/endothelial (CD73, CD105), and hematopoietic (CD43 and CD45)markers, whereas lin⁻ cells lack markers of differentiated hematopoieticcells including CD2, CD3, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16,CD19, and CD20. By day 4, VE-Cadherin⁺ (CD144) cells emerged. Theemerging VE-cadherin⁺ cells represent a heterogeneous population, whichincludes CD144⁺CD235a/43⁻CD73⁺ non-hemogenic endothelial progenitors(non-HEPs), CD144⁺CD73⁻CD235a/43⁻ hemogenic endothelial progenitors(HEPs) and CD144⁺73⁻CD43⁺CD235a⁺CD41a⁻, angiogenic hematopoieticprogenitors. HEPs have the potential to give rise to multipotentlin⁻CD34⁺CD43⁺CD45^(+/−) hematopoietic progenitors.

Unfortunately, the OP9 system relies on mouse feeder cells and serum,which limit its utility for studying hPSC response to specific growthfactors and manufacturing clinical grade therapeutic blood cells. Inaddition, the OP9 coculture system is very sensitive to variations inserum quality, stromal cell maintenance, and size of PSC colonies usedfor differentiation.

Although other investigators have developed feeder-free differentiationprotocols, these protocols rely on forming embryoid bodies (EBs) forhematopoietic differentiation. EB methods often rely on serum and alsohave significant drawbacks, such as asynchronous differentiation andhigh variability. Recently several protocols have been developed toinduce hematopoiesis in serum-free conditions (Salvagiotto, et al.,2011; Wang, et al., 2012); however, they still require xenogeniccomponents, serum albumin, and/or proprietary supplements. It alsoremains unclear whether these protocols reproduce the distinct waves ofhematopoiesis as seen on OP9.

In addition, most protocols differentiate hPSCs grown on MEFs orMatrigel®. Since a new, completely chemically-defined xenogen-freemedium and matrix for hPSC-derivation and maintenance has been described(Chen, et al., 2011), there is a need to develop a similarchemically-defined xenogen-free directed differentiation protocol forderiving hematopoietic progenitors from hPSCs.

Unless defined otherwise in this specification, technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs and byreference to published texts.

It is to be noted that the term “a” or “an,” refers to one or more, forexample, “a molecule,” is understood to represent one or more molecules.As such, the terms “a” (or “an”), “one or more,” and “at least one” areused interchangeably herein. The term “about” as used hereincontemplates a range of values for a given number of +/−10% themagnitude of that number. For example, “about 3 grams” indicates a valueof 2.7 to 3.3 grams, and the like.

As referred to herein, the terms “defined conditions” or “definedmedium” mean the identity and quantity of each ingredient is known. Theterm “ingredient,” as used herein, refers to a component the molecularidentity and quantity of which is known.

Disclosed herein are efficient and reproducible methods and supportingcompositions that recapitulate, in a completely defined, xenogen-freesystem, the hematopoietic development observed in the OP9 co-culturesystem through early mesoderm, hematovascular mesoderm precursor, andhemogenic endothelial stages.

Table 1 provides a list of cell types, associated cell markerphenotypes, and corresponding abbreviations used herein.

TABLE 1 Phenotypic features and definition of subsets with angiogenicand hematopoietic potential from hPSCs presented in the currentapplication Abbreviation Phenotype Designation/Definition PM^(EHM)lin⁻APLNR⁺PDGFRalpha GFR Primitive posterior mesoderm (PM) A⁺P⁺enriched in cells expressing typical primitive streak and lateralplate/extraembryonic mesoderm genes. These cells have potential to formmesenchymoangioblast (MB) and hemangioblast (HB) colonies in serum- freemedium in response to FGF2. HVMP ^(EHM)lin⁻ Hematovascular mesodermalprecursor APLNR⁺KDR^(bright)PDGFRalpha^(low/−) lacking the expression ofprimitive streak genes and highly enriched in bipotentialhematoendothelial cluster forming cells. HEP CD144⁺CD235a/CD43⁻CD73⁻Hemogenic endothelial progenitors that have primary endothelialcharacteristics, lacking hematopoietic CFC potential and surfacemarkers, but are capable of generating blood and endothelial cells uponcoculture with stromal cells. Non-HEP CD144⁺CD235a/CD43⁻CD73⁺Non-hemogenic endothelial progenitors that have all functional andmolecular features of endothelial cells and form endothelial colonies onOP9. AHP CD144⁺CD235a/CD43⁺CD73⁻ Angiogenic blood progenitors thatCD41a⁻ possess primary hematopoietic characteristics and FGF2 andhematopoietic cytokine-dependent colony-forming potential but arecapable of generating endothelial cells. CD43⁺CD235a⁺CD41a⁺Hematopoietic progenitors enriched in erythromegakaryocytic progenitors.lin−CD34+CD43+CD45+/− Multipotential hematopoietic progenitors withmyelolymphoid potentialMethods for Hematoendothelial Differentiation of Human Pluripotent StemCells (hPSCs)

Disclosed herein are methods for the differentiation of humanpluripotent stem cells (either human embryonic or human inducedpluripotent stem cells) under defined conditions, and, preferably, inthe absence of embryoid body formation. At least one desired outcome ofthis differentiation is the provision of endothelial and hematopoieticcell populations that may be a source for functional studies of theselineages as well as a source for clinical therapies.

In some embodiments the differentiation method provided herein includesthe steps of (a) providing human pluripotent stem cells (e.g., humanembryonic stem cells (hESCs) or human induced pluripotent stem cells(hiPSCs)) and (b) culturing the human pluripotent stem cells underhypoxic conditions in a cell culture medium comprising FGF2, BMP4,Activin A, and LiCl for a period of about two days to form a cellpopulation of ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cellswith mesenchymoangioblast potential. Preferably, the human pluripotentstem cells are cultured without formation of embryoid bodies.

In some embodiments, the human pluripotent stem cells are plated at aninitial density of about 5000 cells/cm² to about 15,000 cells/cm², e.g.,6000 cells/cm², 7000 cells/cm², 8000 cells/cm², 9000 cells/cm², oranother plating density from about 5000 cells/cm² to about 15,000cells/cm², In some embodiments, the differentiation method furtherincludes the step of (c) culturing, under hypoxic conditions, the cellpopulation obtained in step (b) in a cell culture medium comprising FGF2and VEGF for a period of about 1-2 days to obtain a cell populationcomprising ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm withhemangioblast (HB-CFC) potential and hematovascular mesoderm cells(^(EMH)lin-KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) enriched in cells with apotential to form hematoendothelial clusters when cultured on OP9 cells.In further embodiments, the differentiation method further includes thestep of (d) culturing the hematovascular mesoderm cells of step (c),under hypoxic conditions, in a cell culture medium comprising FGF2,VEGF, IL6, SCF, TPO, and IL3 for about one day to achieve formation ofCD144⁺CD73⁺CD235a/CD43⁻ non-hemogenic endothelial progenitors (non-HEP),CD144⁺CD73⁻CD235a/CD43⁻ hemogenic endothelial progenitors (HEPs),CD144⁺CD73⁻CD235a/CD43⁺41a⁻ angiogenic hematopoietic progenitors (AHP),and CD43⁺CD41a⁺ hematopoietic progenitor cells.

In some embodiments, the differentiation method further includes thestep of (e) culturing, under normoxia, the HEPs and hematopoieticprogenitor cells in a culture medium comprising FGF2, VEGF, IL6, SCF,TPO, IL3 for about three days to obtain an expanded population of CD43⁺hematopoietic progenitors comprising CD43⁺CD235a+CD41a⁺erythromegakaryocytic progenitors and lin⁻CD34⁺CD43⁺CD45^(+/−)multipotent hematopoietic progenitors.

In some embodiments a differentiation method disclosed herein includesthe step of at least one of: (i) culturing human pluripotent stem cells,without embryoid body formation, under hypoxic conditions in a cellculture medium comprising FGF2, BMP4, Activin A, and LiCl for a periodof about two days to form a cell population of^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells withmesenchymoangioblast potential; (ii) culturing, under hypoxicconditions, ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cellswith mesenchymoangioblast potential in a cell culture medium comprisingFGF2 and VEGF for a period of about 1-2 days to obtain a cell populationcomprising ^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm withhemangioblast (HB-CFC) potential and hematovascular mesoderm cells(EMH^(lin−)KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) enriched in cells with apotential to form hematoendothelial clusters when cultured on OP9 cells;(iii) culturing hematovascular mesoderm cells(^(EMH)lin−KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)) cells, under hypoxicconditions, in a cell culture medium comprising FGF2, VEGF, IL6, SCF,TPO, and IL3 for about one day to achieve formation ofCD144⁺CD73⁺CD235a/CD43⁻ non-hemogenic endothelial progenitors (non-HEP),CD144⁺CD73⁻CD235a/CD43⁻ hemogenic endothelial progenitors (HEPs),CD144⁺CD73⁻CD235a/CD43⁺41a⁻ angiogenic hematopoietic progenitors (AHP),and CD43⁺CD41a⁺ hematopoietic progenitor cells; and (iv) culturing,under normoxia, hemogenic endothelial progenitors (HEPs) and CD43⁺CD41a⁺hematopoietic progenitor cells in a culture medium comprising FGF2,VEGF, IL6, SCF, TPO, IL3 for about three days to obtain an expandedpopulation of CD43⁺ hematopoietic progenitors comprisingCD43⁺CD235a+CD41a⁺ erythromegakaryocytic progenitors andlin⁻CD34⁺CD43⁺CD45^(+/−) multipotent hematopoietic progenitors.

In some embodiments, hypoxic conditions hypoxic conditions refer to alevel of environmental oxygen (e.g., a cell culture incubator gasmixture) of about 3% 02 to about 10% 02. In some embodiments, hypoxicconditions is about 5% 02. In embodiments where a cell culture medium isallowed to equilibrate under hypoxic conditions, the cell culture mediumbecomes a hypoxic cell culture medium due to the lower level ofdissolved oxygen compared to a cell culture medium equilibrated undernormoxic conditions (e.g., a gas mixture containing about 20% oxygen).

In some embodiments, the culture medium to be used in any of theabove-described differentiation methods comprises an IF9S medium, asdescribed herein. In one embodiment, the IF9S medium to be used is theIF9S medium having the formulation set forth in Table 2.

In some embodiments, any of the above-referenced cells (e.g., humanpluripotent stem cells) are cultured on Tenascin C. In some embodiments,any of the referenced cells are seeded on a substrate treated with anamount of Tenascin-C sufficient to adhere 10,000 cells/cm² to thesubstrate. In some embodiments, the Tenascin-C to be used is humanTenascin C. In some embodiments, the substrated is treated with TenascinC at a concentration of at least about 0.25 μg/cm² to 1 μg/cm², e.g.,0.4 μg/cm², 0.5 μg/cm², 0.7 μg/cm², 0.8 μg/cm², or another concentrationfrom at least about 0.25 μg/cm² to 1 μg/cm².

In some embodiments, in the cell culture medium to be used in theabove-described differentiation methods, the concentration of: BMP4 isabout 50 ng/ml to about 250 mg/ml; Activin A is about 10 ng/ml to about15 ng/ml; FGF2 is about 10 ng/ml to about 50 ng/ml; LiCl is about 1 mMto about 2 mM; VEGF is about 20 ng/ml to about 50 ng/ml; SCF is about 50ng/ml to about 100 ng/ml; TPO is about 50 ng/ml to about 100 ng/ml; IL-6is about 50 ng/ml to about 100 ng/ml, and IL-3 is about 5 ng/ml to about15 ng/ml.

In some embodiments, any of the above-referenced cells are cultured in axeno-free cell culture medium. Of central importance for clinicaltherapies is the absence of xenogenic materials in the derived cellpopulations, i.e., no non-human cells, cell fragments, sera, proteins,and the like. Preferably, the present invention arrives at xenogen-freedifferentiated cells by use of Tenascin C or Collagen IV as a platform,which essentially replaces contact with OP9 cells used in earlierdifferentiation systems. In addition, the media disclosed herein arechemically-defined and, in some embodiments, are made xeno-free, andincorporate human proteins, which can be produced using recombinanttechnology or derived from placenta or other human tissues in lieu ofanimal-derived proteins. In some embodiments, all proteins added to themedium are recombinant proteins.

While differentiation processes include ordered, sequential events, thetiming of the events may be varied by at least 20%. For example, while aparticular step may be disclosed in one embodiment as lasting one day,the event may last for more or less than one day. For example, “one day”may include a period of about 18 to about 30 hours. Periods of timeindicated that are multiple day periods may be multiples of “one day,”such as, for example, two days may span a period of about 36 to about 60hours, and the like. In another embodiment, time variation may belessened, for example, where day 2 is 48+/−3 hours from d0; day 4 is96+/−3 hours from d0, and day 5 is 120 hours+/−3 hours from d0.

Examples of potential committed and/or differentiated lineagesobtainable by the present invention include KDR⁺APLNR⁺PDGFRalpha⁺primitive mesoderm cells with HB and MB CFC potential,^(EMH)lin⁻KDR^(hi)APLNR⁺PDGFRalpha^(lo/−) hematovascular mesoderm cellswith potential to form hematoendothelial clusters when cultured on OP9cells, VE-Cadherin⁺ (CD144⁺) subset cells, such as HEPs(CD144⁺CD73⁻CD235a/43⁻, non-HEPS (CD144⁺CD73⁺CD235a/43⁻, and AHPs(CD144⁺CD73⁻CD235a/43⁺41a⁻), CD43⁺ hematopoietic progenitor cells suchas CD43⁺CD235a⁺41a⁺ erythromegakaryocytic hematopoietic progenitors, andlin⁻CD34⁺CD43⁺CD45^(+/−) multipotent hematopoietic progenitors. The termlineage⁻ (“lin⁻), as used herein, refers to a hematopoietic precursor orprogenitor cell that has not have committed to any of its derivativeblood cell lineages as of yet, since it retains the capability todifferentiate into any of them. This characteristic is monitored bylooking for the absence of cell surface markers indicative ofdifferentiation into any of the derivative lineages. A furthersignificant advantage of the present disclosure is the ability to useclonal cell populations due to the Tenascin C platform, which removesreliance on undefined stochastic events common to cell clumps, such asembryoid bodies, to generate differentiated populations. Moreover, theclonal cell populations exhibit greater uniformity during thedifferentiation process, which provides a higher yield of synchronizedcells than previously seen in feeder cell systems. Therefore, thepresent disclosure also describes a more efficient, better scalabledifferentiation system than previously available.

Compositions Defined Cell Culture Media and Concentrated MediaSupplements

Some embodiments herein disclose a differentiation medium comprising abase medium, L-ascorbic acid 2-phosphate Mg²⁺ salt, monothioglycerol,sodium selenite (in addition to any present in the base medium),polyvinyl alcohol, Glutamax™, non-essential amino acids (NEAA),chemically defined lipid concentrate, Holo-Transferrin, and insulin.Suitable base media for the differentiation media described hereininclude, but are not limited to, Iscoves Modified Dulbecco's Medium/F12(IMDM/F12), TeSR1 base medium, which is mTeSR1™ base medium, (Stem CellTechnologies-see Ludwig and Thomson (2007), Curr Protoc Stem Cell Biol.,Chapter 1:Unit 1C.2 and U.S. Pat. No. 7,449,334) without FGF2 andTGF-beta; DF4S base medium, which is Essential 8™ medium (LifeTechnologies; also known as “E8” medium-see Chen and Thomson (2011), NatMethods, 8(5):424-429 and U.S. Patent Application Publication No.20120178166) without FGF2 and TGF-beta, I4S base medium, which is DF4Sbase with Iscove's modified Dulbecco's medium (IMDM) instead ofDMEM/F12, and IF4S base is DF4S base with IMDM/F12 instead of DMEM/F12.Preferably, the base medium to be used is albumin-free. IMDM/F12 is ahighly enriched synthetic medium suited for rapidly proliferating,high-density cell cultures with an added nutrient mixture.

In some embodiments, differentiation media used herein, referred togenerically herein as “IF9S” media, comprises IMDM/F12, L-ascorbic acid2-phosphate Mg²⁺ salt, monothioglycerol, sodium selenite (in addition toany present in the base medium), polyvinyl alcohol, Glutamax™,non-essential amino acids (NEAA), chemically defined lipid concentrate(Life Technologies; Cat. No. 1905031), Holo-Transferrin, and insulin.

In one embodiment, an IF9S medium comprises IMDM/F12 (1×), L-ascorbicacid 2-phosphate Mg²⁺ salt (64 mg/L), monothioglycerol (50 mg/L), sodiumselenite (in addition to any present in the base medium; 8.4 ug/L),polyvinyl alcohol (10 mg/L), Glutamax™ (1×), NEAA (1×), chemicallydefined lipid concentrate (0.1×), Holo-Transferrin (10.6 mg/L), andinsulin (20 mg/L).

As described herein, at various time points/stages of hematoendothelialdifferentiation of hPSCs, the complete differentiation medium to be usedcontains various combinations of cytokines, growth factors, and/or smallmolecules. Depending on the stage of hematoendothelial differentiationaccording to the methods described herein, a suitable completedifferentiation medium will be supplemented with different combinationsof cytokines with concentrations within the ranges described for thecomplete differentiation media described herein.

In some embodiments, complete differentiation medium comprises an IF9Smedium, BMP4, Activin A, FGF2, and LiCl. In other embodiments completedifferentiation medium comprises an IF9S medium, FGF2, and VEGF. Infurther embodiments, complete differentiation medium comprises an IF9Smedium, FGF2, VEGF, SCF, TPO, IL-6, and IL-3. In some embodiments, thefinal complete medium concentration of: BMP4 is about 50 ng/ml to about250 mg/ml; Activin A is about 10 ng/ml to about 15 ng/ml; FGF2 is about10 ng/ml to about 50 ng/ml; LiCl is about 1 mM to about 2 mM; VEGF isabout 20 ng/ml to about 50 ng/ml; SCF is about 50 ng/ml to about 100ng/ml; TPO is about 50 ng/ml to about 100 ng/ml; IL-6 is about 50 ng/mlto about 100 ng/ml, and IL-3 is about 5 ng/ml to about 15 ng/ml. In someembodiments all of the proteins used in the complete differentiationmedium are recombinant human proteins. In other embodiments, thecomplete differentiation medium comprises one or more non-human proteins(e.g., recombinant non-human proteins).

In some embodiments, a complete differentiation medium comprises an IF9Smedium and one of the “cytokine” combinations listed in Table 3 at theindicated concentrations. In some embodiments, the IF9S mediumformulation used in the just-mentioned complete differentiation media isthe IF9S medium formulation set forth in Table 2.

While the presently disclosed media may include the specific morphogens,small molecules, and hematopoietic cytokines disclosed herein, it iscontemplated that additional components with the same, equivalent, orsimilar properties may be used in addition to or in place of thosedisclosed, as are known in the art.

In some embodiments, media disclosed herein may include xenogenic (i.e.,non-human, biologically derived) materials. For example, a xenogenicmaterial may be a recombinant protein of xenogenic origin. Mediadisclosed herein may be also made in concentrated forms that are dilutedprior to use, such as 2×, 10×, 100×, or 1000× concentrations.

Moreover, the replacement of certain xenogenic materials in the media ofthe present invention provided greater, unexpected benefits than justproviding xenogen-free culture conditions. For example, it is believedthat replacement of bovine serum albumin with polyvinyl alcohol led to a“thicker” medium that unexpectedly contributed to cell survival.

TABLE 2 Description of an exemplary embodiment of an IF9S medium. IF9SComponents Concentration IMDM/F12 Base Component L-ascorbic Acid2-Phosphate Mg2⁺ salt 64 mg/L monothioglycerol 40 ul/L additional sodiumselenite 8.4 ug/L polyvinyl alcohol 10 mg/L GLUTAMAX   1× Non-essentialamino acids   1× Chemically defined lipid concentrate 0.1×Holo-Transferrin 10.6 mg/L Insulin 20 mg/L

TABLE 3 Day (hours ± range) (activity) Cytokine Concentration Range O₂Level d0 (0 h) BMP4 50 ng/ml 50-250 ng/ml Hypoxia (change media) ActivinA 12.5 ng/ml 10-15 ng/ml (5% O₂, 5% CO₂) FGF2 50 ng/ml 10-50 ng/ml LiCl2 mM 1-2 mM d2 (48 ± 3 h) FGF2 50 ng/ml 10-50 ng/ml Hypoxia (changemedia) VEGF 50 ng/ml 20-50 ng/ml d4 (96 ± 3 h) FGF2 50 ng/ml 10-50 ng/mlHypoxia (d4) (change media) VEGF 50 ng/ml 20-50 ng/ml d5 (120 ± 3 h) SCF50 ng/ml 50-100 ng/ml Normoxia (d5) (move to normoxic TPO 50 ng/ml50-100 ng/ml (20% O₂, 5% CO₂) incubator) IL-6 50 ng/ml 50-100 ng/ml IL-310 ng/ml 5-15 ng/ml d6 (144 ± 3 h) FGF2 50 ng/ml 10-50 ng/ml Normoxia(add media) VEGF 50 ng/ml 20-50 ng/ml SCF 50 ng/ml 50-100 ng/ml TPO 50ng/ml 50-100 ng/ml IL-6 50 ng/ml 50-100 ng/ml IL-3 10 ng/ml 5-15 ng/mlCytokineSupplement Combinations and Exemplary Concentrations atDifferent Days After Initiating Hematoendothelial Differentiation ofhPSCs (in an IF9S medium).

Concentrated Medium Supplements

Also disclosed herein is a concentrated “9S” medium supplement,comprising L-ascorbic acid 2-phosphate Mg²⁺ salt, monothioglycerol,additional sodium selenite, polyvinyl alcohol, Glutamax™ (or glutamine),non-essential amino acids (NEAA), chemically defined lipid concentrate,Holo-Transferrin, and insulin. In some embodiments, the concentrated 9Smedium supplement comprises each component at a concentration 10× to1000× of the final working concentration once diluted in a base medium.In some embodiments, the concentrations of all of the 9S components inthe concentrated supplement is 10× to 1000× the concentrations listed inTable 3. In some embodiments, the supplement is to be diluted inIMDM/F12 medium to obtain IF9S medium as described herein.

Kits

Also contemplated herein are kits useful for hematoendothelialdifferentiation of hPSCs. In some embodiments, a kit comprises a 9Sconcentrated medium supplement, as described herein, one or more ofBMP4, Activin A, FGF2, LiCl, SCF, TPO, IL-6, and IL-3, and instructionsfor generating an IF9S medium and a method for hematoendothelialdifferentiation of hPSCs as described herein. In some embodiments, a kitfurther includes IMDM/F12 medium. In some embodiments, the kit comprisesa 9S concentrated medium supplement, Activin A, FGF2, LiCl, SCF, TPO,IL-6, IL-3, and instructions for generating an IF9S medium and a methodfor hematoendothelial differentiation of hPSCs as described herein. Infurther embodiments, any of the above-mentioned kits also includeTenascin C (e.g., human Tenascin C), which is used as a substrate foradhesive growth according the differentiation methods described herein.

Defined Cell Culture Systems for Hematoendothelial Differentiation ofhPSCs

Also described herein is a defined cell culture system forhematoendothelial differentiation of hPSCs. Such cell culture systemsinclude a defined differentiation culture medium as described herein,e.g., an IF9S medium, a Tenascin C protein substrate for adherent growthof hPSCs or their differentiated progeny along the hematoendotheliallineage. In some embodiments, Tenascin C is used at at least 0.25 μg/cm²to about 1 μg/cm² to generate a suitable adhesive substrate.

In some embodiments, the cell culture system includes an IF9S mediumsupplemented with BMP4, Activin A, FGF2, and LiCl. In some embodiments,the IF9S medium is supplemented with FGF2 and VEGF. In some embodiments,the IF9S medium utilized in the cell culture system is supplemented withFGF2, VEGF, SCF, TPO, IL6, and IL-3. In some embodiments, the IF9Smedium used in the cell culture system is formulated according to themedium described in Table 2. In some embodiments, the defined cellculture system comprises a cell culture medium that is hypoxic, which isreadily achieved by the use of a cell culture incubator permittingoxygen level regulation, and by equilibrating the cell culture medium ina cell culture incubator set to about 3% O₂ to about 10% O₂ (e.g., 5%O₂).

In further embodiments, the defined cell culture system further includesadherent human pluripotent stem cells cultured on the Tenascin Csubstrate in the IF9S medium according to the methods described herein.

Cells can be grown on, e.g., Tenascin C-coated cell culture dishes,multi-well cell culture plates, or microcarrier beads. Preferably, theTenascin C protein is human Tenascin C (GenBank Accession No.CAA55309.1; available commercially, e.g., Millipore Cat. No. CC065)

The use of Tenascin C and hypoxic conditions enables the generation ofenriched populations of endothelial and hematopoietic cells at higherpercentages than compared to cells seeded on Collagen IV or OP9 cells,such as greater than 10%, or greater than about 20%, greater than about50%, or greater than about 60% when compared per stage per platform (seeFIG. 10 and the Examples). In one embodiment, the percentages of targetpopulations obtainable by the present invention may be greater thanabout 35% for KDR⁺APLNR⁺PDGFRalpha⁺ mesoderm cells or greater than about20% of VE-Cadherin⁺CD43⁻ endothelial cells and greater than about 40% ofCD34⁺CD43⁺ hematopoietic progenitor cells. Further, with respect to FIG.10, the percentages of cells obtained on the Tenascin C platform are theindicated percentage or greater. Further, FIG. 10 represents thepercentage of the target population (e.g., mesoderm progenitor,endothelial progenitor, hematopoietic progenitors with the correspondingphenotypes as according to flow cytometry) of the total culture whendifferentiated on either Col IV or TenC.

The present methods and materials may be combined into cell culturesystems to provide new differentiation platforms. In one embodiment, abasic cell culture system includes pluripotent stem cells seeded onTenascin C. In another embodiment, a cell culture system includes stemcells seeded on Collagen IV, in a medium supplemented with Activin A.These systems have the capacity to produce cell populations enrichedwith hematopoietic progenitor cells.

The cell culture systems contemplated herein may be modular, in thatthey may incorporate additional components that alter the resulting cellpopulations derived from the system. For example, the cell culturesystem may incorporate media that are xenogen-free for certain desiredoutcomes. However, they may include xenogen-containing media if, forexample, clinical therapies are not envisioned for the derived cellpopulations. Further, the cell culture systems may be based on varioussized culture vessels, as are known in the art, to arrive at the desiredcell population production scale.

In some cases, one can substitute some of the components of an IF9Smedium. For example, ascorbic acid and monothioglycerol can be replacedwith an optional supplement of a compound and/or a thiol-containingcompound with antioxidant properties. GLUTAMAX™ can be replaced with anoptional supplement of L-glutamine. “Non-essential amino acids (NEAA),”which is a general term for amino acids that the human body can producefrom other amino acids can be replaced with an optional supplement ofamino acids. “Chemically defined lipid concentrate,” which is a solutionspecifically distributed by Life Technologies, can be replaced with anoptional supplement of lipids. Additional selenite, insulin, andholo-transferrin can be replaced with any ITS supplement. ITS stands for“Insulin-Transferrin-Selenite.” Some companies (e.g., LifeTechnologies), sells ITS solutions to be used as supplements in otherbasal media (DMEM, for example). However, the manufacturer does notprovide concentrations for each component. Polyvinyl alcohol can bereplaced with an optional supplement of a biologically inactive mediathickening compound.

The following examples set forth preferred materials and methods foraccomplishment of the invention. It is to be understood, however, thatthese examples are provided by way of illustration and nothing hereinshould be taken as a limitation upon the overall scope of the invention.

EXAMPLES Example 1 IMDM/F12 Based Media Significantly ImprovesDifferentiation Efficiency of hPSCs into Hematoendothelial Lineage

Previously, our lab developed protocol for the efficient differentiationof hematopoietic hPSC differentiation using a coculture method on themouse stromal cell line, OP9.^(9,13) Although OP9 system supportsefficient generation of HE and multilineage hematopoietic progenitors(FIG. 1), this system is very sensitive to variations in serum quality,stromal cell maintenance, and size of hPSC colonies and clumps used fordifferentiation.^(13,14) Forming embryoid bodies (EBs) is anothercommonly used approach for inducing HE and blood formation fromhPSCs.^(7,15,16) However, EB methods often rely on serum or non-definedmedium and also have significant drawbacks, such as asynchronousdifferentiation, high variability and dependence on initial clump size.Additionally, inconsistency in quality of hPSCs due to variations inalbumin batches used for hPSC maintenance may introduce variations inefficiency of blood production.

To overcome these limitations we decided to identify chemically definedmedium and matrix proteins capable to support hematoendothelialdifferentiation without serum from single cell suspension of H1 humanembryonic stem cells (hESCs) expanded in E8 completely definedxenogene-free medium on vitronectin (VTN)¹⁷.

Methods

Human Pluripotent Stem Cell Maintenance

Human pluripotent stem cells (H1 and H9 hESCs, fibroblast derived iPSC19-9-7T, and BM119-9 iPSCs derived from bone marrow mononuclear cells)were maintained on vitronectin or matrigel in E8 media made in-housesupplemented with FGF2 and TGFβ (Peprotech). Cells were passaged whenthey reached 80% confluency using 0.5 mM EDTA in PBS. The cells weremaintained in normoxic conditions with 5% CO₂.

Human Pluripotent Stem Cell Differentiation

Human pluripotent stem cells were detached from vitronectin or matrigelwhen they reached 80% confluency using 1× TrypLE (Life Technologies) andplated at an optimized density ranging from 5000 cells/cm² to 15,000cells/cm² depending on the cell line onto 6-well plates coated with 0.5μg/cm² of ColIV (Sigma-Aldrich) or 0.5 μg/cm² Tenascin-C (Millipore) inE8 media supplemented with 10 μM Rho Kinase inhibitor (Tocris Y-27632).After 24 hours (day 0), the media was changed to IF9S media supplementedwith 50 ng/ml BMP4 (Peprotech), 15 ng/ml Activin A (Peprotech), 50 ng/mlFGF2 (Miltenyi Biotech), 2 mM LiCl (Sigma), and on occasion, 1 μM RhoKinase inhibitor to increase cell viability. On day 2, the media waschanged to IF9S media supplemented with 50 ng/ml FGF2 and 50 ng/ml VEGF,and 10 μM SB-431542 (Tocris) where mentioned. On day 4, the media waschanged to IF9S media supplemented with 50 ng/ml FGF2, VEGF, TPO, SCF,IL-6, and 10 ng/ml IL-3. On day 6, additional IF9S media supplementedwith the same 6 factors were added to the cultures without aspiratingthe old media (Table 3 Table 3). IF9S (IMDM/F12 with 9 supplements) wasmade in-house with the following: 50% IMDM 50% F12 (Life Technologies)supplemented with 64 mg/L L-ascorbic Acid 2-Phosphate Mg2+ salt(Sigma-Aldrich), 40 ul/L monothioglycerol (Sigma-Aldrich), 8.4 μg/Ladditional sodium selenite (Sigma-Aldrich), 10 mg/L polyvinyl alcohol(Sigma-Alderich), 1× glutamax (Life Technologies), 1× non-essentialamino acids (Life Technologies), 0.1× chemically defined lipidconcentrate (Life Technologies), 10.6 mg/L Holo-Transferrin(Sigma-Aldrich), and 20 mg/L Insulin (Sigma-Aldrich) (Table 2).Differentiation was conducted in hypoxic condition from day 0 to day 5,and transferred to normoxic condition from day 6 to day 9 (FIG. 1). The1× TrypLE was used to dissociate and collect cells for analysis.

Mesenchymo- (MB) and Hemangioblast (HB) Assay

MB and HB were detected using serum-free CFC medium supplemented withFGF2 assay as previously described¹¹. Day 2 or 3 cultures weredissociated and prepared in a single-cell suspension using 1× TrypLE(Life Technologies) and 5,000 cells of the total culture were platedinto the CFC media. MB and HB colonies were scored 12 days after platingthe single-cell suspension.

Hematopoietic CFC Assay.

Hematopoietic CFC were detected using serum-containing H4436 Methocult™supplemented with human recombinant SCF, G-CSF, GM-CSF, IL-3, IL-6, andEPO (Stem Cell Technologies). Hematopoietic potential of AHPs wasevaluated using serum-free SF H4236 methocult with added FGF2 (20ng/ml), SCF (20 ng/mL), IL3 (10 ng/mL), IL6 (10 ng/mL), and EPO (2 U/mL)(Stem Cell Technologies) as previously described⁶. 1000-10000differentiated cells were plated into the CFC medium and the colonieswere scored after 14 days of culture.

Flow Cytometry and FACS

Flow Cytometry was conducted using the using a FACSCalibur flowcytometer and following antibodies: CD31-FITC (clone WM59), CD34-FITC(8G12), CD41a-FITC/APC (clone HIPS), CD43-FITC/PE/APC (clone 1G10),CD45-APC (clone HI30), CD73-FITC/PE (clone AD2),CD144-FITC/PE/AlexaFluor647 (clone 55-7H1), CD235a-FITC/PE/APC (cloneGA-R2), KDR-PE/AlexaFluor647 (clone 89106), PDGFRα-PE (clone aR1) (BDBiosciences), TRA-1-85-FITC/PE (clone TRA-1-85), and APLNR-APC (clone72133) (R&D Systems). Sorting was conducted on a FACS Aria, as describedpreviously 46. The purity of isolated populations was 92-95%.

Secondary Culture of Differentiated hPSCs onto OP9

OP9 cells were maintained in α-MEM (Gibco) supplemented with 20% FBS(Hyclone) as previously described.¹⁰ Sorted day 4 or day 5 cultures wereplated on a confluent layer of OP9 cells in α-MEM (Gibco) supplementedwith 10% FBS (Hyclone) supplemented with 100 μM MTG, 50 μg/ml ascorbicacid, 50 ng/ml SCF, TPO, IL-6, and 10 ng/ml IL-3 at a density of 5,000cells/well of a 6 well plate as previously described⁶. Cultures wereprepared for flow cytometry 4 to 7 days later by collecting floatingcells and dissociating the entire cultures using 1× TrypLE.

T-Cell Differentiation of Day 9 Cultures

An OP9 cell line (OP9-DLL4) constitutively expressing human delta-likeligand 4 (DLL4) was established by our lab using lentivirus and wasmaintained similarly to OP9. After human pluripotent stem cells weredifferentiated for 9 days, the floating cells were collected, strainedthrough a 70 μm cell strainer (BD Biosciences) and washed. Then, theywere resuspended in T-cell differentiation media consisting of α-MEM(Gibco) supplemented with 20% FBS (Hyclone) supplemented with IL7 (5ng/ml), Flt3L (5 ng/ml) and SCF (long/ml). Then, they were plated on anOP9-DLL4 and cultured at 37° C. and 5% CO2. After 4 days, the cells wereharvested using collagenase IV (Gibco) solution (1 mg/ml in DMEM/F12,Gibco) and 1× TrypLE (Invitrogen), and passaged onto a fresh layer ofOP9-DLL4. After 3 days, the cells are passaged again. Subsequentpassages are conducted every 7 days up to 4 weeks, after which thefloating cells are collected for flow analysis and genomic DNAextraction for TCR rearrangement assay.

TCR Rearrangement Assay

Genomic DNA was isolated using quick-gDNA MiniPrep (Zymo Research). TCRβand TCRγ clonality was detected using a PCR amplification kit(Invivoscribe) and AmpliTaq Gold® DNA polymerase (Applied Biosystems) aspreviously described³³. The PCR products were analysed usingheteroduplex analysis on a 6% polyacrylamide gel stained with ethidiumbromide.

Microarray Analysis of Mouse Stromal Cell Lines

A mouse bone marrow stromal cell line, OP9, was obtained from Dr. ToniNakano (Research Institute for Microbial Diseases, Osaka University,Japan), S17 was obtained from Dr. Kenneth Dorshkind (University ofCalifornia, Los Angeles) and MS-5 was obtained from the German TissueCulture Collection. Stromal cell lines were cultured as described⁹.DNA-free RNA was isolated using RiboPure™ RNA and DNAse using TURBO™DNAfree reagents (Ambion). All samples were processed at the GeneExpression Center of the Biotechnology Center at the University ofWisconsin, Madison and analyzed using A4543-00-01 MM8 60mer expr Musmusculus 1-Plex Array standard arrays manufactured by NimbleGen Systems(Madison, Wis.). Gene expression raw data were extracted usingNimbleScan software v2.1. Considering that the signal distribution ofthe RNA sample is distinct from that of the gDNA sample, the signalintensities from RNA channels in all eight arrays were normalized with aRobust Multiple-chip Analysis (RMA) algorithm⁴⁷. Separately, the samenormalization procedure was performed on those from the gDNA samples.For a given gene, the median-adjusted ratio between its normalizedintensity from the RNA channel and that from the gDNA channel was thencalculated as follows: Ratio=intensity from RNA channel/(intensity fromgDNA channel+median intensity of all genes from the gDNA channel). Geneswith more than 3 fold differences in expression were considereddifferentially expressed. Only genes with expression level >1 wereselected for analysis.

Results

IMDM/F12 Based Medium Significantly Improves Differentiation Efficiencyof hPSCs into the Hematoendothelial Lineage

We plated hESCs as single cells and allowed to attach over 24 hours inE8 media supplemented with 10 μM Rho kinase inhibitor on Matrigel (MTG),VTN, or Collagen IV (ColIV). Then, the media was changed to one of threebasal media free of animal proteins, or growth factor-free TeSR1,supplemented with human recombinant BMP4, FGF2, and VEGF factors whichcommonly used to induce blood formation from hPSCs^(18,19). After 4 daysof differentiation cell cultures evaluated for the presence ofKDR^(hi)CD31⁺ cells which are highly enriched in hematoendothelialprogenitors⁶. Flow cytometry analysis showed that cells differentiatedon ColIV-coated plates in IMDM/F12 media supplemented with L-ascorbicAcid 2-Phosphate Mg²⁺ salt, 8.4 μg/L additional sodium selenite,Holo-Transferrin, and insulin differentiated most efficiently intoKDR^(hi)CD31⁺ hematoendothelial precursors (FIG. 8). Later, we foundthat the addition of polyvinylalcohol, NEAA, Glutamax, chemicallydefined lipid concentrate, and monothioglycerol increased cell viabilityand differentiation efficiency (data not shown). The subsequent basalmedia is referred to as IF9S (IMDM/F12 plus 9 supplements). Theseresults demonstrated that the selected media and supplements made itpossible to obtain hematoendothelial cells in a chemically defined,xenogene-free conditions on ColIV matrix from hPSCs maintained in E8media.

Example 2 Analysis of a Unique Molecular Signature ofHematopoiesis-Supportive Stromal Cells Identified Tenascin C as anExtracellular Matrix that Promotes the Development and Maintenance ofHematopoietic Precursors

Previously, we showed that OP9 is superior to other stromal cell linessuch as S17, and MS5 in induction of hematopoietic differentiation⁹. Itwas also found that day 8 overgrown OP9 cultures are superior to day 4freshly confluent OP9 in induction of hematopoietic-CFCs, includingmultipotential GEMM-CFCs⁹. Since the confluency of the stromal cellsaffect differentiation efficiency, this led us to believe that there isan extracellular matrix influencing hematoendothelial differentiation.In order to find the matrix protein(s) critical forhematopoiesis-supportive activity of OP9 we performed molecularprofiling of S17 and MS5 stromal cell lines with lowhematopoiesis-inducing potential and OP9 cells. In addition, we comparedovergrown OP9 (day 8) with freshly confluent OP9 (day 4) monolayers.Transcriptome analysis revealed 21 genes differentially expressed in day8 overgrown OP9 cells as compared to all other stromal cells (FIG. 2a ).These included genes encoding Ptn (pleiotrophin), a secreted regulatorof HSC expansion and regeneration,²⁰ Rspo3 (R-spondin 3), an importantregulator of Wnt signaling and angioblast development²¹, and anextracellular matrix proteins Postn (periostin) required for Blymphopoiesis,²². Interestingly, one gene that showed the mostsignificant expression change in overconfluent OP9 was Tnc (Tenascin C)(FIG. 2b ). TenC is expressed in mesenchymal cells underlyinghematopoietic clusters in the Aorta-Gonado-Mesonephros (AGM) region andis required for intraembryonic and postnatal hematopoiesis²³⁻²⁵. It isalso expressed in the bone marrow stem cell niche²⁵. Because of theseunique properties, we tested whether TenC could support hematopoieticdifferentiation more effectively than ColIV.

Example 3 Time- and Dose-Dependent Treatment of FGF2, BMP4, Activin a,LiCl, and VEGF Induces Mesodermal, Endothelial, and Hematopoietic Stagesof Development

Our prior studies identified distinct stages of hematoendothelialdevelopment following hPSC differentiation in coculture with OP9 (FIG.1)^(6,9-11,26). Plating hPSCs onto OP9 stromal cells induces formationof primitive streak and mesodermal cells which can be detected based onexpression apelin receptor (APLNR) and KDR (VEGFR2)¹¹ and lack ofexpression Endothelial (CD31, CD144 (VE-cadherin)),endothelial/Mesenchymal (CD73, CD105) and Hematopoietic (CD43, CD45)markers, i.e. by ^(EMH)lin− phenotype. The first KDR⁺ mesodermal cellsappearing in OP9 coculture on day 2 of differentiation express APLNR andPDGFRalpha (^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ hereafter referred as A⁺P³⁺cells). These cells display mesenchymoangioblast (MB) potential, i.e.capacity to form colonies with both mesenchymal stem cell (MSC) andvascular potential. On day 3 of differentiation A⁺P³⁺ cells acquireblast (BL)-CFC or hemangioblast (HB) potential¹¹. Both MB and HBpotentials can be detected using colony-forming assay in serum-freeclonogenic medium supplemented with FGF2¹¹. With advanced maturation,mesodermal cells loss BL-CFC activity and upregulate KDR expression anddownregulate PDGFRalpha, i.e. acquire KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)hematovascular progenitor (HVMP) phenotype which enriches in cells withthe potential to form hematoendothelial clusters on OP9⁶. Theendothelial stage of development is defined by expression ofendothelial-specific marker VE-cadherin (CD144). The first VE-Cadherin⁺(CD144⁺) cells emerge from KDR^(hi)APLNR⁺PDGFRalpha^(lo/−) mesodermalcells by day 4 of differentiation. The emerging VE-cadherin⁺ (CD144+)cells represent a heterogeneous population which include CD43⁻CD73⁺(CD144⁺CD43⁻CD73⁺) non-hemogenic endothelial progenitors (non-HEPs) andCD43⁻CD73⁻ (CD144⁺CD43⁺CD73⁻) hemogenic endothelial progenitors (HEPs)⁶.HEPs lacking hematopoietic CFC potential, but acquire it after culturewith stromal cells. The hematopoietic stage of development is defined byexpression of hematopoietic-specific marker CD43^(6,10). The first CD43⁺cells emerge within VE-cadherin⁺ (CD144+) cells on day 4-5 ofdifferentiation. These cells express low level CD43 and coexpressCD235a, but lack CD41a expression, i.e. had CD144⁺CD43/235a⁺41a⁻phenotype. Because these cells have capacity to form hematopoieticcolonies in presence of FGF2 and hematopoietic cytokines as well to growendothelial cells on fibronectin, we designated them as angiogenichematopoietic progenitors (AHPs). The first CD41a cell appears withinCD235a positive cells. CD235a⁺CD41a⁺ cells are highly enriched anderytho-megakaryocytic progenitors and lacking endothelial potential. Theprogenitors with broad myelolymphoid potential andlin⁻CD34⁺CD43⁺CD45^(+/−) phenotype can be detected in hPSC culturesshortly after emergence of CD235a⁺CD41a⁺ cells. Acquisition of CD45expression by lin⁻ cells is associated with progressive myeloidcommitment.¹⁰

To reproduce the hematoendothelial program observed in OP9 coculture wedecided to select the optimal combinations of morphogens for mesoderminduction and hematoendothelial specification and define specific growthfactors required for step-wise progression of differentiation toward HEand blood cells in hPSC cultures (FIG. 1) differentiated inchemically-defined conditions on ColIV and TenC. During embryonicdevelopment, BMP4, Wnt, and TGFβ/Nodal/Activin A signaling have beenfound to be critical to initiate primitive streak formation andsubsequent mesoderm development^(27,28). It has been shown thatactivation of these signaling pathways is essential to induce theexpression of brachyury and KDR (Flk-1, VEGFR2), and initiate mesodermalcommitment of mouse and human PSCs^(18,19,29-32). We have found thathigh concentrations of BMP4 (50 ng/ml) combined with low concentrationsof Activin A (15 ng/ml) and a supplement of LiCl (2 mM) consistentlyinduced expression of the mesodermal surface markers APLNR, KDR, andPDGFRalpha after 2 days of culture of single cell suspension of hESCs onColIV in chemically-defined conditions as we described above. However,these conditions poorly supported cell survival and required theaddition of FGF2 and a hypoxic conditions (5% O₂, 5% CO₂) to improvecell viability and output of mesodermal cells. Day 2 KDR⁺ mesodermalcells differentiated in these conditions expressed APLNR and PDGFRalpha,i.e. became APLNR⁺PDGFRalpha⁺ cells and displayed MB colony-formingpotential similar to APLNR⁺PDGFRalpha⁺ mesodermal cells obtained fromday 2 hPSCs differentiated in OP9 coculture¹¹ (FIG. 3). After 2 days ofdifferentiation, we found that only FGF2 and VEGF are required forAPLNR⁺PDGFRalpha⁺ mesoderm to acquire HB potential on day 3 ofdifferentiation and advance mesoderm specification toward HVMPssignified by increase in KDR expression and the decrease in PDGFRalphaexpression in APLNR⁺ cells (KDR^(hi)APLNR⁺PDGFRalpha^(lo/−) phenotype)in CD31⁻ mesodermal cells on day 4 of differentiation. The pattern ofdevelopment was similar in cells cultured on ColIV and TenC, however thelater one produced significantly higher APLNR⁺PDGFRalpha⁺ cells, MB andHB colonies (FIG. 3a, 3c ).

Day 4 differentiated hESCs lost capability to form HB colonies (FIG. 3c), however these cells were capable to form hematoendothelial clusterswhen sorted and plated onto OP9 in αMEM supplemented with 10% FBS, SCF,TPO, IL6, and IL3. The hematoendothelial cluster potential wasrestricted to KDR^(hi)APLNR⁺PDGFRalpha^(lo/−)CD31⁻ HVMPs (FIG. 3d ). TheKDR^(lo)CD31⁻ cells only formed endothelial clusters with almost nohemogenic activity (FIG. 3d ), while KDR− cells fail to grow both,endothelial and blood cells (not shown). This is also consistent withdifferentiation in OP9 coculture⁶. The percentage ofKDR^(hi)APLNR⁺PDGFRalpha^(lo/−) HVMPs cells was consistently higher inTenC cultures (FIG. 3a ).

Because formation of HVMPs in hPSC/OP9 cocultures is closely followed bydevelopment of HE and blood progenitors, we supplemented our cultureswith SCF, TPO, IL-6, and IL-3 hematopoietic cytokines in addition toVEGF and FGF2 starting from day 4 of differentiation. Although wenoticed that the continuous treatment of cultures with FGF2 and VEGF wassufficient for induction of endothelial progenitors and hematopoieticspecification, addition of hematopoietic cytokines was essential toincrease output of these cells in chemically defined cultures. On day 5of differentiation in these conditions, the previously identified 3major subsets of the CD144⁺ populations⁶ emerged: CD144⁺CD43⁻CD73⁺,CD144⁺CD43⁻CD73⁻ and CD144⁺CD43/CD235a⁺CD41a⁻ (FIG. 4). When thesesubsets were sorted and plated into endothelial conditions, all of themformed a monolayer of VE-cadherin expressing cells with capacity touptake AcLDL and form vascular tubes in the tube formation assay,consistent with OP9 coculture (FIG. 4d ). However, hematopoietic CFCpotential was mostly restricted to CD144⁺CD43/CD235a⁺CD41a⁻ cells (FIG.4c ). Importantly similar to finding with day 5 CD144⁺ subsets generatedin coculture with OP9, the hematopoietic CFC potential ofCD144⁺CD43/CD235a⁺CD41a⁻ cells was detected only in serum-free medium inpresence of FGF2 in addition to hematopoietic cytokines, indicating thatthese cells essentially similar to AHP identified in hPSC/OP9coculture⁶. We previously defined HEP as CD144⁺CD43⁻CD73⁻ cells lackinghematopoietic CFC potential, but capable to acquire it after culture onOP9. To determine whether CD144⁺CD43⁻CD73⁻ generated in completelydefined conditions similar to OP9-induced HEPs, we sorted day 5 CD144⁺subsets and cultured with OP9 as previously described⁶. In theseconditions, the HEPs formed both endothelial and hematopoietic cellswith large number of HE-clusters, while AHPs formed predominantlyhematopoietic cells with few endothelial cells and hematoendothelialclusters. CD144⁺CD43⁻CD73⁺ cells formed endothelial clusters onlyconsistent with non-HEP phenotype (FIG. 4d ). Cultures differentiated onTenC had a larger population of total CD144⁺ cells, thereby increasingthe population of HEPs, non-HEPs, and AHPs compared to culturesdifferentiated on ColIV (FIG. 4a, b ).

When numerous floating round hematopoietic cells became visible incultures on day 6, the hypoxic conditions were not necessary to sustainhematopoietic development. Therefore, from day 6 of differentiation, thecultures were transferred cells to a normoxic incubator (20% O₂, 5%CO₂). By day 8 of differentiation, cultures showed development of largenumber of CD43+ hematopoietic cells composed CD235a⁺CD41a⁺ cellsenriched in erythro-megakaryocytic progenitors and lin⁻CD43⁺CD45^(−/+)cells which expressed CD34 (FIG. 5) and lacked of other lineage markers(not shown). Consistent with cells differentiated on OP9, hematopoieticcolony forming potential was limited to the CD43⁺ subpopulations (FIG.5c ). CD43⁺ hematopoetic progenitors expanded significantly more on TenCcompared to ColIV (FIG. 5b ). In addition, the GEMNI-CFC potential ofcultures on TenC was significantly greater than cultures on ColIV (FIG.5d ).

Although the differentiation protocol was initially developed using H1hESCs, we found that chemically defined conditions described here alsosupports formation of HE and blood from other hESCs (H9) and hiPSCsgenerated from fibroblasts or bone marrow mononuclear cells (FIG. 9).Previously, we found that hiPSC obtained through reprogramming of cordblood mononuclear cells (CB hiPSCs) differentiate less efficiently intothe blood cells on OP9 feeders compared to fibroblast-derived (FB)hiPSCs³³. These findings have been reproduced when we differentiated CBand FB iPSCs on ColIV. However, differentiation on TenC restoredhematopoietic differentiation potential of CB hiPSCs to the level seenwith hESCs and FB hiPSCs (FIG. 9), thereby confirming that TenC issuperior over ColIV in promoting hematopoietic differentiation fromhPSCs.

Example 4 Tenascin C Uniquely Supports Specification of T LymphoidProgenitors from hPSCs

To find out whether our culture system supports establishment ofdefinitive hematopoietic program from hPSCs, we analyzed T cellpotential of blood cells generated in our system as indicator ofdefinitive hematopoiesis⁷. When we collected CD43⁺ floating cells fromday 9 differentiated cultures, and replated them onto OP9 cellsexpressing DLL-4 in α-MEM with 20% FBS, Flt3L, IL-7, and SCF, CD7⁺CD5⁺lymphoid progenitors began to emerge by week 2 of secondary coculture.By week 3, CD4⁺CD8⁺ double positive T-cells arose (FIG. 6a ).

Interestingly, CD43⁺ cells generated on both ColIV and TenC matrices hada capacity to generate CD5⁺CD7⁺ lymphoid progenitors. However,progression toward CD4⁺CD8⁺ T lymphoid cells was observed only fromCD43⁺ cells generated on TenC but not on ColIV. To confirm T celldevelopment, we analyzed genomic DNA from these cultures for thepresence of TCR rearrangements. This analysis demonstrated the presenceof multiple PCR products of random V-J and D-J rearrangements at theγ-locus and multiple V-J and rearrangements at the γ-locus indicative ofpolyclonal T lineage repertoire (FIGS. 6b and 6c ). Overall, thesefindings signify that extracellular matrix Tenascin C is essential forsupporting definitive hematopoiesis in completely chemically definedconditions.

Example 5 Inhibition of TGF-β Promotes Hematoendothelial Specificationin Chemically Defined Conditions

Recent studies have shown that adding TGF-β inhibitors after mesodermspecification but before endothelial development increases definitivehematopoietic differentiation. We found that when 10 μM of SB-431542, apotent but non-specific TGFβ inhibitor, is added from day 2 to day 4, itsignificantly decreases the development of PDGFRalpha-positive mesodermcells by day 3, and increases CD31⁺ differentiation by day 4. After day4, SB-431542 is no longer added, but the effect of the 2 day treatmentcontinues to increase CD43⁺ population by day 9 (FIG. 7).

During the last decade significant progress has been made inhematopoietic differentiation from hPSCs. Multiple protocols forhematopoietic differentiation have been developed and made it possibleroutinely produce blood cells for experimentation. However, generationof blood cells with long-term reconstitution potential, HSCs, from hPSCsremains significant challenge. In the embryo, hematopoietic cells andHSCs arise from specific subset of endothelium (HE)¹⁻⁵, thus the abilityto interrogate signaling pathways leading to HE specification andtransition into the blood cells in completely chemically definedenvironment is essential for identification of factors required for HSCspecification and eventually development of conditions for de novo HSCgeneration. Although original protocols for hematopoieticdifferentiation have employed xenogenic, feeder and/or serum, severalserum- and feeder-free systems for hematopoietic differentiation havebeen described recently^(18,34,35) However, these protocols stillrequires serum components (albumin), and it remains unclear whetherthese protocols reproduce distinct waves of hematopoiesis, includinggeneration of HE with definitive lymphomyeloid potential, observed inthe original differentiation systems. More recently Kennedy et al,⁷ havedeveloped feeder- and stroma-free conditions for EB-based hematopoieticdifferentiation of hPSCs and showed that these conditions reproducedprimitive and definitive waves of hematopoiesis and generate HE with Tlymphoid potential. However, this protocol uses hPSCs growing on MEFsfor EB-based hematopoietic differentiation in proprietary medium withnon-disclosed chemical and human protein content. Here we developed forthe first time protocol that enable efficient production of blood cellsin completely chemically defined conditions free of serum and xenogeneicproteins from a single cell suspension of hPSCs maintained in chemicallydefined E8 medium¹². This protocol eliminates variability associatedwith animal- or human-sourced albumins, xenogenic matrix, clump sizesand asynchronous differentiation observed in EB system and reproducestypical waves of hematopoiesis, including formation of HE and definitivehematopoietic progenitors, observed in hPSCs differentiated on OP9.Importantly, based on molecular profiling of OP9 and stromal cell lineswith different hematopoiesis-inducing activity, we found that TenCmatrix protein uniquely expressed in OP9 with robust hemato-inducingpotential, strongly promotes hematoendothelial and T lymphoiddevelopment from hPSCs. TenC is disulfide-linked hexameric glycoproteinthat is mainly expressed during embryonic development. Although TenCmostly disappear in adult organism, its expression upregulated duringwound repair, neovascularization and neoplasia.³⁶ Interestingly TenC isfound in adult bone marrow where it expressed predominantly in endostealregion^(37,38) and upregulated following myeloablation²⁵. TenC supportsproliferation of bone marrow hematopoietic cells³⁹ and erythropoiesis⁴⁰.TenC-deficient mice had lower bone marrow CFC potential²⁴, failed toreconstitute hematopoiesis after bone marrow ablation and showed reducedability to support engraftment of wild type HSCs²⁵. High level of TenCexpression was also detected in human and chickenaorta-gonad-mesonephros (AGM) region^(23,41), the site where the firstHSC emerge, and hematopoietic sites in the human fetal liver⁴². BecauseTenC expression is highly enriched in subaortic mesenchyme rightunderneath of hematopoietic clusters, it was suggested that TenC playspivotal role in HSC development during embryogenesis²³. TenC is alsoinvolved in regulation of angiogenesis and cardiac endothelialprogenitors⁴³. Our studies demonstrated the superior properties of TenCin promoting hematopoiesis from hPSCs. The positive effect of TenC wasobvious at all stages of differentiation and included the enhancement ofhemogenic mesoderm, HE and CD43⁺ hematopoietic progenitors production.Importantly, TenC was able to support development of definitivehematopoietic cells with T lymphoid potential, while we were not able toobtain such cells in cultures on ColIV. TenC molecule is composed of anamino-terminal oligomerization region followed by heptad repeats,EGF-like and fibronectin type III repeats and fibrinogen globe³⁶. Thefunction of these domains is poorly understood. It is believed thateffect and interaction of TenC with cells requires the integrate actionof multiple domains⁴⁴, although several unique mitogenic domains capableof inducing a proliferation of hematopoietic cells were identifiedwithin this molecule³⁹. Several signaling mechanisms implicated in cellinteraction with TenC have been identified, including suppression offibronectin-activated focal adhesion kinase- and Rho-mediated signalingand stimulation of Wnt signaling (reviewed in⁴⁵). Further studies aimedto identify mechanism of TenC action on hPSCs and their hematopoieticderivatives would be of value to understand the role of this matrixprotein in hematopoietic development.

In summary, the findings provided here identified TenC matrix proteinsand completely chemically defined conditions free of serum/serumcomponents and animal proteins capable of supporting the scalableproduction of HE and definitive blood cells from hPSCs. Thisdifferentiation system allows precise interrogation of signalingmolecules implicated in hematopoietic differentiation and provideplatform for production of cGMP grade of blood cells for clinicalapplication.

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We claim:
 1. A xenogen-free, albumin-free culture system fordifferentiating human pluripotent stem cells into mesoderm, endothelialand hematopoietic progenitor cells comprising: a) a solid substratecomprising a layer of Tenascin C or collagen and seeded with humanpluripotent stem cells; and b) xenogen-free and albumin free culturemedium comprising: about 50 to about 250 mg/ml BMP4; about 10 to about15 ng/ml Activin A; about 10 to about 50 ng/ml FGF2; and about 1 toabout 2 mM LiCl, wherein the culture system produces^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells.
 2. Thexenogen-free, albumin-free culture system of claim 1, wherein the mediumfurther comprises a base medium, L-ascorbic acid 2-phopshate Mg2+ slatmonothioglycerol, sodium selenite, polyvinyl alcohol, non-essentialamino acids (NEAA), chemically defined lipid concentrate,Holo-transferrin, and insulin.
 3. The xenogen-free, albumin-free culturesystem of claim 2, wherein the medium comprises 64 mg/L L-ascorbic acid2-Phosphate Mg²⁺ salt, 40 μl/L monothioglyverol, 8.4 μg/L additionalsodium selenite, 10 mg/L polyvinyl alcohol, 1× nonOessential aminoacids, 0.1× chemically-defined lipid concentrate, 10.6 mg/Lholo-transferrin, and 20 mg/L insulin.
 4. The xenogeny-free culturesystem of claim 1, wherein the medium of step b further comprises: about50 to about 100 ng/ml SCF, about 50 to about 100 ng/ml TPO, about 50 toabout 100 ng/ml IL-6, and about 5 to about 15 ng/ml IL-3.
 5. Thexenogen-free, albumin-free culture system of claim 1, wherein the humanpluripotent stem cells are seeded at a concentration of at least about0.25 μg/cm² to about 1 μg/cm².
 6. The xenogen-free, albumin-free culturesystem of claim 1, wherein the solid substrate comprises a layer ofTenascin C or collagen.
 7. The xenogen-free, albumin-free culture systemof claim 6, wherein the layer of Tenascin C is at a concentration of atleast about 0.25 μg/cm² to about 1 μg/cm².
 8. A xenogen-free culturesystem for differentiating human pluripotent stem cells, comprising: a.^(EMH)lin⁻KDR⁺APLNR⁺PDGFRalpha⁺ primitive mesoderm cells seeded on asolid substrate comprising Tenascin C or collagen; and b. mediumsupplemented with about 10 to about 50 ng/ml FGF2; and about 20 to about50 ng/ml VEGF.
 9. The xenogen-free culture system of claim 8, the mediumof (b) further comprising hematopoietic cytokines.
 10. The xenogen-freeculture system of claim 9, wherein the hematopoietic cytokines comprise:about 50 to about 100 ng/ml SCF, about 50 to about 100 ng/ml TPO, about50 to about 100 ng/ml IL-6, and about 5 to about 15 ng/ml IL-3.